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Frequently Asked Questions

On this page you will find answers to frequently asked questions (FAQs) about this website and product line. If your question is not answered here, please contact our Technical Support Staff, a team of experienced scientists based in Rockford, IL, our U.S. manufacturing site for Thermo Scientific Pierce Protein Research Products.

  1. What happened to the ABR-Affinity BioReagents name?
  2. Why has the website appearance changed?
  3. What is the best way to find and select an antibody on this website?
  4. How should I store a supplied antibody to maximize shelf-life?
  5. Western blotting problems?
  6. ELISA problems?
  7. Immunohistochemistry (IHC) problems?
  8. Immunoprecipitation (IP) problems?

What happened to the ABR-Affinity BioReagents name?

In 2008, ABR-Affinity BioReagents was acquired by Thermo Fisher Scientific Inc. Now sold as the technology brand Thermo Scientific, the entire portfolio of existing ABR-Affinity BioReagents products have been integrated in operations and support with the Thermo Scientific Pierce Protein Research product line. In time, the ABR-Affinity BioReagents name will no longer be used.

 

Why has the website appearance changed?

As we continue to integrate our offering of antibody-related products under the Thermo Scientific Technology brand, we continue to improve the functionality of our website to provide better search- and filtering- capabilities and more relevant connectivity to related products and resources. Significant changes in appearance will continue throughout 2009 and 2010 as we implement these improvements. We welcome your suggestions; please contact Technical Support.

 

What is the best way to find and select an antibody on this website?

  1. Use the search box at the upper right corner of the page.
  2. Type the name of the target or protein of interest (e.g., "caspase" or "caspase 3")
    or type the product number (with or without dashes, e.g., "MA1-7664" or "MA17664").
  3. Filter the search results using one of the features listed in the left margin (e.g., "By Class" or "By Host")
  4. Click a column heading to sort the search results according to data in that column.

If a target name returns no results, try alternative spellings, abbreviations or synonyms. If a product number returns no results, try using only the last four or five digits of the number that follow the dash (e.g., "7664"). If you still cannot find a product or are unsure how to choose among the many available option, please do not hesitate to contact Technical Support for assistance.

 

How should I store a supplied antibody to maximize shelf life?

Briefly:

  1. Store antibodies frozen (-20°C) in lyophilized or concentrated liquid form until needed. Avoid repeated freeze/thaw cycles, which degrade proteins. Instead, freeze reconstituted antibodies in single-use aliquots or add anti-freeze (glycerol or ethylene glycol) so that the stock solution can be maintained at -20°C in liquid form to enable pipetting without "thawing".
  2. Avoid storing antibody (or other protein solutions) for more than a few days at 4°C. Concentrated stocks (> 0.5mg/mL) are usually stable at 4°C for at least 2-3 weeks if they are sterile-filtered and contain anti-microbial additives, such as sodium azide. Do not attempt to store diluted (< 0.1mg/mL) antibody solutions unless stabilized by addition of "carrier" proteins or special additives.
  3. Do not use frost-free freezers. These units subject the freezer compartment to regular freeze/thaw cycles to eliminate the frost on the sides of the freezer. These cycles will degrade protein solutions.

For more information, see our list of Tech Tips and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products. In particular, see Tech Tip #43: Protein Stability and Storage.

 

Western Blot problems?

Common antibody-based causes of Western blotting problems:

  1. The antibody being used is not suitable for Western blot procedures, which generally require antibodies that are capable of binding reduced and denatured target proteins. Make certain that WB is listed as a validated application for the antibody.
  2. Primary and secondary antibody concentrations are not correct (too concentrated, which is especially common, or too dilute). Test several combinations of dilutions to optimize conditions. Refer to the Tech Tip listed below.
  3. The secondary antibody is too specific or not specific enough for the species and subclass of the primary antibody. Refer to the Tech Tip listed below.
  4. The primary or secondary antibody is degraded or inactive as a result of improper storage, improper dilution or excessive freezing and thawing. Include positive control lanes or blots to ensure that the antibodies are working properly. Refer to the Tech Tip listed below.

For more information, see our list of Tech Tips and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products. In particular, see the following documents:

  1. Tech Tip #67: Chemiluminescent Western blotting technical guide and protocols
  2. Tech Tip #22: Determine source of nonspecific background signal in Western blots
  3. Tech Tip #24: Optimize antigen and antibody concentrations for Western blots
  4. Tech Tip #59: Choosing a secondary antibody - a guide to fragment specificity
  5. Article: Overview of Western Blotting

 

ELISA problems?

Common causes of high background:

  1. Insufficient washing – Wash according to the protocol. Rapidly fill wells with wash solution using a squirt bottle; then immediately to dump out the liquid, and then tap the inverted plate on an absorbent tissue. Repeat at least three times.
  2. Incorrect incubation times – Follow set protocol.
  3. Contamination – Use the highest-quality available distilled/deionized water for diluting the stock reagents.

Common causes of weak signal or no color development:

  1. Improper storage.
  2. Reagents have expired.
  3. Wavelength-plate may have been read at the wrong wavelength.

For more information, see our list of Tech Tips and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products. In particular, see the following resources:

  1. Tech Tip #33: Guide to enzyme substrates for ELISA
  2. Article: Overview of ELISA

 

Immunohistochemistry (IHC) problems?

Common causes of weak, diffuse or no signal:

  1. The antibody being used is not suitable for IHC procedures. Make certain that IHC is listed as a validated application for the antibody.
  2. The fixation procedure (using formalin and paraformaldehyde fixatives) might be masking the epitope that the antibody recognizes. Try using antigen retrieval methods to unmask the epitope.
  3. The primary and/or secondary antibody may be bad due to improper storage, improper dilution or excessive freezing and thawing. Be sure to run positive controls to ensure that the primary and/or secondary antibody is working properly.
  4. Deparaffinization may be insufficient. Deparafinize the sections longer and change out the xylene.
  5. The protein is located in the nucleus and the antibody cannot penetrate the nucleus. Add a permeabilizing agent to the blocking buffer and antibody dilution buffer.
  6. The PBS buffer may be contaminated with bacteria that damage the phosphate groups on the protein of interest. Add 0.01% azide in the PBS antibody storage buffer or use fresh, sterile PBS.
Common causes of high background in IHC:
  1. The blocking serum used might be incorrect. Be sure to block according to the provided protocol and references.
  2. Blocking might be insufficient. Increase the blocking incubation period to overnight at 4°C.
  3. The primary antibody concentration being used is too high. Be sure to titrate the antibody to determine the optimal concentration.
  4. The secondary antibody might be binding non-specifically. Make certain to run a secondary-only control.
  5. Pre-absorb primary and secondary antibodies with 5% normal serum from the host species of the secondary antibody.
  6. Endogenous peroxides may be active. Add a quenching step to the protocol (hydrogen peroxide).

For more information, see our list of Tech Tips and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products.

 

Immunoprecipitation (IP) problems?

Common causes of high background in IP procedures:

  1. The antibody concentration being used is too high. Be sure to titrate the antibody to determine the optimal concentration.
  2. There is non-specific binding of other proteins to the affinity matrix being used. Be sure to preclear samples with affinity matrix before performing IP experiments.

Common causes of target protein not being precipitated?

  1. The antibody being used may not be suitable for immunoprecipitation procedures. Make certain that IP is listed as a validated application for the antibody.
  2. The antibody being used may not be able to detect the species being tested. Make certain that the species being testing is listed as a validated target for the antibody.
  3. The washing solution is too harsh. Use a milder (less stringent) wash buffer. For example, if you had been using RIPA buffer, which contains SDS and reducing agents, try using simple PBS instead.
  4. The antibody species and subclass does not bind to the affinity resin being used. Make certain that the antibody being used is compatible with the particular affinity matrix being used (e.g., Protein A or Protein G bead type).

For more information, see our list of Tech Tips and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products. In particular, see the following resources:

  1. Tech Tip #64: Immunoprecipitation technical guide and protocols.
  2. Tech Tip #34: Binding characteristics of Protein A, Protein G, Protein A/G and Protein L
  3. Products: Thermo Scientific Pierce IP and Co-IP Kits